Cancer cells and many cells of genetic diseases have alterations in the cell cycle. The basic understanding of the normal cell cycle is therefore a key step for understanding of the disease processes and thus finding eventual cure. Chromosome replication is a crucial event in the cell cycle. Escherichia coli adjusts the overall rate of chromosome replication, as required when growing in different growth conditions, by precisely regulating the frequency of initiation of rounds of chromosome replication. Thus, the mechanism and regulation of the initiation event has been a focus of intense studies for a number of years. E. coli has two other initiation mechanisms for chromosome replication which are normally repressed but can be activated under certain specific conditions. One mechanism, which is activated in SOS-induced cells, depends on homologous recombination functions and is thought to utilize a D-loop for a site of initiation. The other mechanism, which can be seen in rnhA mutants deficient in RNase H activity, is proposed to utilize an R-loop for an initiation site. The long range of this research project is to characterize these initiation mechanisms at the molecular level. It is hypothesized that the D-loop dependent replication is involved both in homologous recombination and in double-strand break repair. Several experiments including density-label experiments designed to directly test the hypothesis are proposed. A working model how R-loops are generated in the cell is also proposed. The model hypothesizes that a transcript RNA strand invades duplex DNA catalyzed by the RecA recombinase and that RecG protein, a helicase known to catalyze branch migration in the homologous recombination process, opposes this RecA-catalyzed reaction. Experiments are proposed to test several key aspects of this working model. These experiments are expected to yield important information about how RNase H functions in the cell. Such knowledge may be vital to the effort to develop antisense DNA oligonucleotides as antiviral and anticancer agents. In addition, this proposed research addresses the classic mystery: Why is the combination of a recA and a polA mutation lethal? An answer to the question is expected to contribute to our understanding of the inter- dependence of the DNA replication and homologous recombination processes.